Project Abstract The overall goal of this project is to design, develop and commercialize compact cryocooling instruments for fast, reproducible and reliable cryopreservation of biomolecular crystals for X-ray cryocrystallography. X-ray crystallography is the most powerful and widely used tool for determining the molecular structures of proteins, viruses, nucleic acids and biomolecular complexes. These structures are critical to modern molecular biology and to the development of pharmaceutical treatments for conditions and diseases. Plunge cooling crystals in liquid nitrogen and collecting data at T=100 K simplifies storage, transport and handling, and dramatically increases the amount of X-ray data that can be obtained from each crystal. However, the cooling process itself damages crystals. Many important targets including viruses, membrane proteins and biomolecular complexes can be very difficult to successfully cool. Moreover, current cooling methodologies, which are time-consuming and done by hand, lead to large variability in cooling outcomes and diffraction quality, and may not accurately capture all salient details of the room/biological temperature molecular structure. The proposed instruments will provide reliable, semi-automated cooling at user adjustable rates, and so provide maximum control and flexibility in designing and implementing cooling protocols. Cooling rates of up to 80,000 K/s, 100 times larger than in current best practice, will eliminate crystalline ice formation during cooling and dramatically reduce required cryoprotectant concentrations. Entry-level, high-throughput and research instruments will be developed using a modular platform, as well as fast-response thin-film temperature sensors needed to measure the large cooling rates these instruments will deliver. Studies of how crystal properties and cooling protocols interact in determining X-ray diffraction outcomes will validate the utility of these instruments and establish protocol for their most effective use. These instruments will significantly improve the efficiency and efficacy of biomolecular crystallography pipelines, and provide new capabilities to academic, government and industrial scientists working to advance our understanding of the molecular mechanisms of life and disease.